This proposal is submitted in response to RFA-RM-13-022, entitled Revisions to Add Single Cell Analysis to Active Research Projects (R01). The studies represent a joint effort from the laboratories of Oleg Denisenko and Diane Krause to apply novel single cell epigenetic assays to biological questions in megakaryopoiesis addressed in the parent grant. We propose the development and application of a powerful novel technology to interrogate chromatin structure at the single cell level. Specifically, we will use the new methods that Dr. Denisenko is developing to probe changes in the epigenetic state of individual genes as primary human megakaryocyte-erythroid progenitor (MEP) cells undergo megakaryocyte lineage commitment and differentiation. The ability to analyze the epigenetic state in single cells has been a roadblock in our studies of hematopoietic specification and differentiation. The aims represent progressive steps 1) from the development of the single cell epigenetic assay in human hematopoietic cell lines to primary human cells; 2) from analysis of the EGR1 locus (which the Denisenko laboratory has already successfully imaged) to the ARH-GEF2 locus, which is the focus of the parent R01; and 3) from analysis of changes in the densit of abundant histone modifications to more specific analysis of transcription factor binding. By applying this novel single cell approach, these studies will not only advance the aims of the parent R01, but will also provide feedback to Dr. Denisenko on the further development of his approach. Analysis of single cell epigenetic changes in primary human megakaryocyte-erythroid progenitor cells and megakaryocytes at different stages of maturation will complement the aims of the parent R01 and bring us closer to the longterm goal of determining the basic molecular mechanisms underlying specification and maturation of megakaryocytes. Such analyses will provide insight into the percentage of cells undergoing specific epigenetic modifications at a given gene, will allow assessment of whether the changes are monoallelic versus biallelic, and the kinetics of such changes at the single cell level compared to other epigenetic modifications and gene expression levels, again all at the level of single cells. These studies, which will be performed over a 2 year period, will position us for future studies focused on how disruptions in these processes cause hematological diseases.